Method to reduce the physiologic effects of drugs on mammals

ABSTRACT

A method to reduce the physiologic effects of drugs in vivo by inducing specific anti-drug antibodies using drugs conjugated to carrier molecules so as to reduce a drug&#39;s toxicity and its physiologic effects upon the recipient. This method includes the treatment and prophylactic prevention of drug abuse, specifically for cocaine and nicotine, and to help reduce the toxic effects of drugs, such as anti-neoplastics.

FIELD OF THE INVENTION

This invention relates to a method for reducing the physiologic effectsof drugs on mammals in vivo. Specifically, the method of this inventionconsists of inducing specific anti-drug antibodies in mammals withimmunogens consisting of a drug conjugated to a carrier molecule. Moreparticularly, the method of this invention consists of generatingantibodies towards addictive substances—particularly cocaine andnicotine—such that the physiologic effects of such addictive substancesare reduced. As a result, toxicity to such addictive substances isdramatically lowered in those mammals which have boon treated with theimmunogens prior to introduction of the addictive substance. Inaddition, mammals can be afforded prophylactic protection from suchaddictive substances by the administration of the drug-conjugatedimmunogen prior to introduction of the addictive substance.

BACKGROUND OF THE INVENTION

The National Institute of Drug Abuse National Household Survey on drugabuse estimated that in 1991 there were approximately 12 million personsin the United States that abused drugs. This included approximately 4.5million occasional abusers of cocaine and greater than 800,000 habitualusers of cocaine. The United States government is projected to spend anestimated $12 billion in 1993 on federal drug control programs with anestimated $2.7 billion allocated to help defray the cost of drugtreatment programs. Typical methods of drug treatment therapy includepsychological counseling. Heroin addiction is sometimes treated withmethadone therapy to permit gradual withdrawal, however, the replacementdrug methadone is itself addictive.

It would be highly desirable to develop a method to neutralize orminimize the effects of controlled substances or addictive drugs therebyrendering continued abuse of addictive substances unproductive due tothe reduced physiologic effect of the drug on the user. Additionally, aphysiologic reduction of the action of the drug, or reduction of therate in which the drug effects the mammalian subject, would helpcontribute to reduce the toxicity of the drug. Such a method would behighly desirable in the treatment of habitual substance abusers, in theprophylactic prevention of abuse, to reduce toxicity of drugs, and mayprovide an additional method to slowly release toxic anti-neoplasticdrugs.

SUMMARY OF THE INVENTION

The present invention consists of the administration of an immunogenconsisting of a drug conjugated to a carrier molecule. The immunogeninduces in the recipient the production of antibodies to the drug, aswell as to the carrier molecule in most cases. Such antibodies, in turn,sequester a subsequently administered drug thereby reducing the drug'sphysiologic effects in the recipient. To demonstrate the method of thisinvention, an anti-cocaine immunogen was synthesized that markedlydemonstrated a reduction in the physiologic effects and toxicity ofcocaine in laboratory animals. A “reduction in the physiologic effect ofa drug” is defined herein as that which can be readily observed andmeasured, such as heart rate, breathing rate, auditory stimulus, eyedilation, irritability, agitation, and pain reflex, in the case ofcocaine.

The present invention describes a method for preventing or treating drugabuse by administering drug-conjugated immunogen that induces in therecipient antibodies, which specifically recognize and neutralize atargeted controlled substance. Such antibodies are then available in thetreated recipient to reduce or eliminate the effect of a subsequentintake of the drug including reduction of the physiologic effects ofsubsequent drug use and reduction of the toxicity of the drug. Theinvention therefore is of great assistance in drug treatment programs.

The method of this invention may also be employed to reduceanti-neoplastic drug toxicity and to provide for a method of slow invivo release of drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immunoblot showing reactivity of cocaine-conjugatedimmunogens with antisera of an animal treated with the anti-cocaineimmunogen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The drug-conjugated immunogen of the present invention is a substancewhich induces in a mammal the production of antibodies which minimize orneutralize the effect of a targeted controlled substance or drug.Antibodies against addictive substances, drugs, or anti-neoplasticcompounds also reduce the toxic effects of the respective drug bykeeping the blood level of unbound drugs in the bloodstream at arelatively low level while the drug is cleared by enzymes, thereticulo-endothelial system, the liver, secreted via the kidneys, orother methods used in mammals to dispose or clear toxic substance. Theability of the drug-conjugated immunogen to reduce such toxic effectsthereby minimizes the likelihood of a drug overdose. Toxicity resultingin death, seizure and tissue damage is greatly diminished thereby.

A controlled substance or drug is defined herein to include thosesubstances known to be subject to abuse, e.g., cocaine, morphine(opium), heroin, and tetrahydrocannabinol (marijuana). Additionalsubstances included in the invention are those which are habit formingsuch as various pain relievers, stimulants, anti-depressant drugs andnicotine. These substances, as well as other drugs which are potentialcandidates for abuse or that are toxic, are contemplated as targets forthe drug-conjugated immunogen and method of the present invention, andare thus to be included in the definition of “controlled substances” or“drugs” for purposes of the present invention.

Anti-cocaine immunogens and a method to treat cocaine abuse areexemplified herein, but it is understood that the immunogen and methodof the present invention may similarly be applied to other controlledsubstances or drugs. The method of the invention reduces the toxicity ofdrugs—due to antibody sequestering of the target drug induced by thedrug-conjugated immunogen—and thus provides a method of releasing toxicdrugs, such an anti-neoplastic compounds, into the bloodstream of thehost mammal, thereby slowly reducing the side-effects of toxic compoundsor drugs.

Cocaine, most controlled substances, and anti-neoplastic drugs arecompounds having low molecular weights and do not tend to elicit aneffective immune response when injected into mammals. However, cocaine,controlled substances, and anti-neoplastic compounds may be conjugatedto carrier molecules. The resulting conjugate is of sufficient size andthus becomes immunogenic so as to generate an immune response, therebyproducing antibodies against the drug of the conjugate.

Carrier molecules suitable for conjugation with controlled substancesand drugs that would generate an immunogen include, for example, sheepalbumin, polysaccharide such as mannan, and various lipopolysaccharidessuch as those derived from Salmonella typhosa. Many conventionalcarriers known to those skilled in the art may also be used in thisinvention, including those approved by regulated governmental agenciesfor use in humans, such as, for example: Diphtheria, Tetanus, andPertussis vaccines or components thereof; poliovirus vaccines andcomponents thereof; Rubella, Mumps, and Measles vaccines or componentsthereof; Hepatitis vaccines (A,B,C, and delta) and components thereof;Haemophilus (A and B) vaccines and components thereof; vaccinia andsmallpox vaccines and components thereof; varicella-zoster vaccines andcomponents thereof, as well as synthetic multiple antigenic peptides(MAPs) and derivatives thereof.

The conjugation method used will depend upon the chemistry of coupling aparticular drug to a particular carrier. Suitable agents and methods forconjugating a variety of compounds are commercially available fromPierce Chemical Co. (Rockford, Ill.), as described, for example in thePierce Chemical Co. catalog. Known methods for conjugating two or morecompounds via specific reactive groups may be applied to the preparationof the drug-carrier molecule conjugates of the present invention. Ingeneral, a reactive site on a first compound is linked to a reactivesite on a second compound, using a coupling agent or catalyst.

Coupling agents include EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCL, (Pierce Chemical Company, Catalog No. 22980),glutaraldehyde and other similar agents. (See, for example, Axen et al.,Nature, 214:1302-1987; and Okawa et al., Journal of ImmunologicalMethods, 149:127, 1992).

The compound conjugated to the carrier may be a natural or syntheticdrug, controlled substance, anti-neoplastic compound, or a derivativethereof. For example, the cocaine derivative benzoylecgonine was used toform a drug-carrier conjugate which induces the production of antibodiesagainst cocaine in a recipient.

The drug-carrier conjugate may contain a drug to carrier ratio of 1:1 orgreater, e.g., 75:1, depending upon available conjugation sites on thecarrier molecule. In some cases, a polymeric carrier may be used whichmay carry a large number of drug molecules, e.g., greater than 100:1drug:carrier.

The drug-conjugated immunogens of the present invention are immunogenic.A particular drug-conjugated immunogen may be tested for immunogenicity,for example, by immunization of test animals and analysis of theresulting antisera. A drug-conjugated immunogen may be modified to makethe composition more immunogenic. Such modifications includesconjugation to lipopolysaccharides, synthetic peptides, or to moleculeswhich stimulate the immune system.

The drug-carrier conjugate immunogens of the present invention areadministered to a recipient by known, conventional methods, e.g.,injection, inhalation, or by oral delivery, depending upon thephysiological characteristics of the drug and carrier compounds.Preferably, the agent is administered by subcutaneous injection. Theconjugate is prepared in a pharmaceutically acceptable carrier, forexample, in in aqueous medium such as water, buffer, saline, glycine, oran oil based carrier, as appropriate for the specific drug-carrierconjugate and the desired mode of delivery.

A therapeutically effective dose of the drug-conjugated immunogen isadministered to a recipient mammal, e.g., human, rabbit, monkey ormouse. A therapeutically effective dose of the drug-conjugated immunogenis one that induces in the recipient the production of antibodies to thedesired drug or controlled substance, which antibodies are effective toreduce or eliminate a response to a subsequent challenge or intake ofthe controlled substance, anti-neoplastic agent, or drug. It isunderstood that a single administration or multiple administrations ofthe drug-conjugated immunogen may be required to induce adequateantibody titres, depending upon each recipient's immune competence. Ingeneral, one to four injections will be used.

Successful immunization of the recipient may be monitored, for example,by analyzing the ability of a recipient's anti-serum to bind thecontrolled substance or drug of choice. Immunoassay methods such asELISA and immunoblot may be used for such analysis. Immunized mammals,when presented with a challenge dose of controlled substance or drug,exhibit diminished effects or no effect of the controlled substance ordrug.

The method of the present invention may be used to immunize mammals,including rabbits and humans, against controlled substances and otherdrugs as a method of preventing or treating drug abuse or to reduce adrug's toxic effects. Immunization against such drugs as cocaine,heroin, opium, morphine, marijuana, nicotine, and anti-neoplasticcompounds should reduce or nullify the physiologic effects of thesedrugs in the recipient and thereby help to reduce the drug's toxicity.

The invention may be better understood by reference to the followingexamples:

EXAMPLE 1 Preparation of a Benzoylecgonine-Conjugated Immunogen

To form a conjugate between benzoylecgonine, a cocaine derivative, andsheep albumin, 5 mg of sheep albumin was conjugated to 1 mg ofbenzoylecgonine using 100 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl, Pierce Chemical Company, Catalog No. 22980) in 1 ml of0.1 M MES buffer (2-(N-morpholino)ethanesulfonic acid) at pH 4.5 forfour hours at 50° C. The mixture was dialyzed against water for two daysat room temperature using a 1,000 m.w. cutoff dialysis tubing.Approximately 5 mg of the benzoylecgonine-albumin conjugated immunogen(COALB) in a volume of 1.5 ml was recovered and used in the subsequentstudies.

Similarly, conjugates of benzoylecgonine to mannan or lipopolysaccharidewere formed by dissolving 10 mg of mannan (Sigma Chemical Co. #M7504) orSalmonella typhosa lipopolysaccharide (Sigma Chemical Co. #L6386) in 0.1M sodium carbonate buffer, pH 10.7, to which 10 mg of cyanobromide(CNBr) was added in a volume of 1 ml. The mixture was allowed to reactfor ten minutes at room temperature. Diaminopropane (50 μl) was addedand the mixture was allowed to sit overnight at room temperature. Thisprocess provided amine groups on the polysaccharide for coupling ofbenzoylecgonine. The mixture was dialyzed with water overnight using a1000 molecular weight cutoff dialysis membrane to yield activatedpolysaccharide. To 5 mg of the activated polysaccharide in 0.9 ml volumewas added 5 mg of benzoylecgonine in 50 μl of 30% ethanol followed bythe addition of 50 mg of EDC in 50 μl of 0.5 M MES buffer at pH 4.5.This solution was incubated at 50° C. for a period of four hours thendialyzed against water for 24 hours using 1000 molecular weight cutoffdialysis membrane. Approximately 5 mg of each conjugate,benzoylecgonine-mannan (COMAN) and benzoylecgonine-lipopolysaccharide(COLPS) in a volume of 1.5 ml was recovered and used in the subsequentstudies.

EXAMPLE 2 Conjugation of Benzoylecgonine to Diphtheria Toxin

Similarly benzoylecgonine can be coupled to other carrier molecules suchas Diphtheria Toxin. In this example, 5 mg. of Diphtheria toxin (SigmaD7544) is dissolved in 0.9 ml of 1.0M MES (pH 4.5) to which is added 1mg. of benzoylecgonine dissolved in a volume of 0.05 ml. 0.1M MES (pH4.5) and to which 10 mg. EDC in 0.05 ml of 0.1M MES (pH 4.5) is added.This coupling reaction is performed at 50° C. for four hours then themixture is dialyzed against water or PBS using a 1000 molecular weightcut-off dialysis membrane or purified by passing the mixture over an gelfiltration column. The recovered conjugate can then be used asdrug-conjugated immunogen.

EXAMPLE 3 Preparation of Anti-Neoplastic-Conjugated Immunogens

Anti-neoplastic drugs, or other highly toxic drugs, may be directlycoupled to carrier molecules as a method to lower the toxicity of thedrug and to provide controlled release or degradation of the drug. Forexample, methotrexate can also be coupled to albumin, diphtheria toxoid,tetanus toxoid, and modified polysaccharide as described above. In thisexample, 5 mg. of a carrier molecule, which has amine groups forbinding, such as tetanus toxoid, is coupled to 1 mg. of methotrexate in0.9 ml of 0.1M MES (pH 4.5) to which is added 10 mg. of EDC dissolved in0.1 ml of 0.1M MES (pH 4.5). The mixture is allowed to couple theanti-neoplastic, or other toxic substance, to the carriermolecule—tetanus toxoid in this example—for four hours at 50° C. afterwhich the drug-conjugated immunogen is purified by dialyzing againstwater or a buffered solution, such as PBS, or is purified by columnfiltration or by High Pressure Liquid Chromatography. The resultingdrug-conjugated immunogen elicits an immune response in a mammal therebygenerating antibodies against the conjugated agent:methotrexate.

EXAMPLE 4 Immunization With Benzoylecgonine-Conjugated Immunogen

Two rabbits were immunized with the cocaine-conjugated immunogen COALB,prepared as described for Example 1. Rabbits were immunized with COALBby subcutaneous injection of 100 μg of the benzoylecgonine-conjugatedimmunogen every two weeks for a period of two months (four injections).Freund's complete adjuvant was used in the first injection and Freund'sincomplete adjuvant was utilized for subsequent injections.

To test for the presence of anti-cocaine antibodies, the serum of thetreated rabbits was analyzed by immunoblot assay for its ability to bindthe benzoylecgonine conjugates produced in Example 1.

Approximately 1 μg of each drug-carrier conjugated immunogen was spottedonto nitrocellulose strips and allowed to air dry. These strips wereblocked for one hour at room temperature using 1:10 Mega-block I (ONASCOProducts, Houston, Tex.).

Sera obtained from the test rabbits both before and after immunizationwith COALB were diluted 1:100 in the blocking solution and applied tothe test strips. After incubating for two hours at room temperature, thestrips were washed four times in a solution of saline and 0.01% tween-20and the secondary antibody was added (1:1000 dilution of goatanti-rabbit antibody conjugated with alkaline phosphatase, in 1:100Mega-block I blocking buffer). The strips were incubated for two hoursat room temperature then washed four times in 0.01% tween-20 solution.Bound antibodies were detected by reacting the substrate NBT/BCIP(Promega Biotech) with the bound secondary antibody which resulted in avisually detectable signal.

The results of this immunoblot assay are shown in FIG. 1, where strip Ashows rabbit sera prior to immunization and strip B shows rabbit seraafter four doses of the COALB. The COMAN (1), COLPS (2) and COALB (3)immunogens each reacted with antibody present in the rabbit sera afterimmunization. The background signal in the pre-immunization COALB (3)spot is expected due to the nature of the albumin carrier.

The immunized rabbits were then challenged with an intravenous dose ofcocaine-HCl (Sigma chemical Co., #C5776) in sterile saline at 2 mg/kg wt(LD₅₀ of 12 mg/kg). Control rabbits challenged with the same dose ofcocaine showed marked grande maul convulsions, involuntary eyemovements, rapid and shallow breathing, were unresponsive to bothvisual, auditory, and pain stimuli, had dilated eyes, but recoveredafter 10-15 minutes. Clearly, the i.v. administered cocaine causedpronounced physiologic effects upon the animals and nearly caused thedeath of one of the animals due to the drug's toxic effects upon therabbit. Two rabbits immunized with the COALB immunogen, when injectedwith the same dose of cocaine (2 mg/kg), showed none of the dramaticeffects that the control rabbits had demonstrated. The treated animalsremained responsive, and no physiologic effects of the cocaine injectionwere noted, which clearly demonstrated that the anti-cocaine immunogen,COALB, was effective in suppressing the physiologic effects of thecocaine challenge as well as apparently lowering the drugs toxicity asdemonstrated by the lack of physiologic trauma when compared to anun-immunized animal.

EXAMPLE 5 Immunization With Benzoylecgonine-Conjugated Immunogen

In a similar fashion, anti-benzoylecgonine immunogens described above inExample 2 (those conjugated to diphtheria or tetanus toxoid) are used toimmunize mammals other than rabbits (such as monkeys and humans) tosuppress the action of cocaine and its derivatives upon the centralnervous system and to increase tolerance of the mammal to the drug. Onehundred μg of the anti-cocaine immunogen is absorbed to an approvedadjuvant aluminum hydroxide and is injected s.c. Similarly,anti-neoplastic or anti-toxic conjugates could be used asdrug-conjugated immunogens in this example.

EXAMPLE 6 Preparation of a Nicotine-Conjugated Immunogen

Nicotine (3-1-Methyl-2-pyrrolidinyl)pyridine;1-methyl-2-(3-pyridyl)pyrrolidine;beta-pyridyl-alpha-N-methyl-pyrrolidine) is conjugated to a carriermolecule (albumin, cationized albumin, amine-linked mannan, amino-linkedpolysaccharide, diphtheria toxoid, tetanus toxoid, or other suchmolecules) using the PharmaLink Immunogen Kit commercially availablefrom Pierce chemical company, Rockford, Ill., catalog number 77158 G.Following the instructions provided, nicotine-HCl is dissolved in theconjugation buffer which includes the carrier compound, SuperCarrier.The coupling agent provided in the Pierce kit is then added and themixture in incubated for 2 to 24 hours at approximately 37-57° C. (Thetime required is related to the temperature of incubation.) Theresulting conjugate is then purified from non-conjugated nicotine usinga desalting column.

Alternatively, approximately 1 mg of carrier protein, e.g. tetanus ordiphtheria toxoid, is dissolved in 200 μl of 0.1 M MES buffer, pH 4.5,0.15M NaCl, and approximately 1 mg. of nicotine-HCl in 200 μl of 0.1 MMES buffer, pH 4.5, 0.15 M NaCl is added. A volume of 50 μl of 37%formaldehyde is added next and the mixture is allowed to react at 37° C.for approximately 3 hours. The solution is dialyzed overnight againstwater using a 1000 m.w. cutoff membrane.

The resulting nicotine-carrier conjugated immunogen is then used toimmunize a recipient, following the procedures for Example 2.

EXAMPLE 7 Immunization With Nicotine-Conjugated Immunogen

Humans and monkeys are immunogenized with anti-nicotine immunogenswherein the conjugated immunogen is absorbed onto a pharmaceuticallyapproved adjuvant aluminum hydroxide and injected i.m. using a dosewhich causes the generation of relatively high titre antibodies (100μg-500 μg). Several injections may be required before suitable antibodytitres are obtained.

In a similar fashion, toxic compounds, such as anti-neoplastic agents,are coupled to carrier molecules using the same method of coupling via acondensation reaction to active hydrogen molecules. One mg. of a carriermolecule, such as sheep albumin, is dissolved in 0.2 ml. of 0.1 M MES(pH 4.5) and 0.15 M NaCl. to which 1 mg. of vinblastine dissolved in 0.2ml of ethanol is added followed by the addition of 0.05 ml. of 37%formaldehyde. The condensation-coupling reaction is allowed to proceedat 37° C. for 3 hours after which the mixture is dialyzed, or otherwisepurified, to remove the salts, buffering agents, formaldehyde andunbound vinblastine. The immunogen is then used to generate antibodiestowards vinblastine by immunizing a mammal (mouse, rabbit, monkey, orhuman) so as to lower the drug's toxicity during treatment in the samemanner as described above.

EXAMPLE 8 Preparation of Morphine-Conjugated Immunogen

Morphine (7,8-Didehydro-4,5-epoxy-17-methyl-morphinan-3,6-diol) isconjugated to albumin, mannan, or lipopolysaccharide using conjugationmethods as described for Examples 1 and 6. Approximately 5 mg ofmorphine acetate-trihydrate (Sigma Chemical Co.) is dissolved in 0.5 mlof 0.1 M MES, pH 4.5. To this is added approximately 5 mg of sheepalbumin dissolved in 0.45 ml of 0.1M MES, pH 4.5. EDC, 50 mg in 50 μl of0.1 M MES, pH 4.5 is added and the mixture reacted for approximately 4hours at 50° C. The resultant conjugate is dialyzed against waterovernight at room temperature, changing water approximately every 2hours and using a 1000 m.w. cutoff membrane.

The resultant morphine-conjugated immunogen is used to immunize mammals(rabbits, mice, monkeys, or humans) as described in Example 2. Theresulting antibodies to morphine sequester morphine, and derivativesthereof, thereby suppressing its effects upon the central nervous systemand thereby reducing the toxicity of the drug.

1.-20. (canceled)
 21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a drug conjugated immunogen, wherein the drug is cocaine or a derivative thereof, and the drug is conjugated to a carrier which is capable of eliciting an immune response.
 22. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a drug conjugated immunogen, wherein the drug is selected from the group comprising of heroin, opium, morphine, marijuana, or a derivative thereof, and the drug is conjugated to a carrier which is capable of eliciting an immune response.
 23. The pharmaceutical composition of claim 21 or 22, wherein the drug is conjugated to a bacterial toxoid.
 24. The pharmaceutical composition of claim 21 or 22, which is suitable for oral, inhalation or injection administration to a human.
 25. The pharmaceutical composition of claim 21 or 22, which is suitable for subcutaneous administration to a human.
 26. The composition of claim 21 or 22, further comprising aluminum hydroxide.
 27. A method of treating or preventing cocaine abuse in humans, the method comprising administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a drug conjugated immunogen, wherein the drug is cocaine or a derivative thereof, and the drug is conjugated to a carrier which is capable of eliciting an immune response.
 28. A method of treating or preventing abuse of a controlled substance in humans, the method comprising administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a drug conjugated immunogen, wherein the drug is selected from the group comprising of heroin, opium, morphine, marijuana, or a derivative thereof, and the drug is conjugated to a carrier which is capable of eliciting an immune response.
 29. The method of claim 27 or 28, wherein the drug is conjugated to a bacterial toxoid.
 30. The method of claim 27 or 28, wherein the composition is administered orally, by inhalation or by injection.
 31. The method of claim 27 or 28, wherein the composition is administered subcutaneously.
 32. The method of claim 27 or 28, wherein the composition further comprises aluminum hydroxide. 